Synthesis of internally fluorescence-quenched sugar nucleotides

Article abstract of DOI:10.1055/s-2004-829563

The synthesis of intramolecularly quenched UDP-Gal analogues bearing a fluorescence emitter attached to the galactose moiety and a quencher on the uracil portion is described. The rate of transfer using several galactosyltransferases was examined. Our res

Full text of DOI:10.1055/s-2004-829563

1784
LETTER
Synthesis of Internally Fluorescence-Quenched Sugar Nucleotides
S
ynthesis of In
r
ternally
a
F
luorescen
n
c
e-Quenched
k
Sugar
N
ucleotide
S
s
chweizer,*a,b Ole Hindsgaul,*a Monica M. Palcica
a
Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada
b
Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
Fax +1(204)4747608; E-mail: schweize@ms.umanitoba.ca; E-mail: hindsgaul@crc.dk
Received 7 April 2004
linkage, which is cleaved during the course of the
enzymatic reaction. A variety of donor/quencher pairs
such as 5-(2-aminoethyl)aminonapthalene-1-sulfonic
acid (Edans)/4-(4-dimethylaminophenylazo)benzoic acid
(Dabsyl)8a and o-aminobenzamide (ABz)/3-nitrotryrosine
[Tyr(NO2)]8b have been previously described to have ideal
spectroscopic properties for internal fluorescence quench-
ing. This paper describes the synthesis of the UDP-Gal
sugar nucleotide analogue 1 bearing an emitter (Abz) on
the galactose moiety and a quencher [Tyr(NO2)] on the
uracil portion (Figure 1). The sugar nucleotide analogues
2 and 3 were also prepared. We designed the modified
sugar nucleotide analogue 1 based on the following ratio-
nale: a) both fluorophores are linked to the sugar nucleo-
tide via short linkers to ensure maximal fluorescence
quenching; b) the Abz-fluorophore is linked to the 6-posi-
tion of galactose since it was expected that modifications
at the 6-positon would have the best chance of retaining
enzymatic activity; c) the quencher is linked to the 5-pos-
iton of uridine since 5-modified uridine analogues are
well documented in the literature.9 A different approach,
in which energy transfer between the fluorophores on
acceptor and the donor sugar was used to monitor a
sialyltransferase catalyzed reaction, has recently been
reported.18
Abstract: The synthesis of intramolecularly quenched UDP-Gal
analogues bearing a fluorescence emitter attached to the galactose
moiety and a quencher on the uracil portion is described. The rate
of transfer using several galactosyltransferases was examined. Our
results demonstrate that galactose-modified, sugar-nucleotide-mod-
ified and double modified UDP-Gal analogues are recognized as
weak substrates by three of the galactosyltransferases.
Key words: carbohydrates, nucleotides, glycosylations, enzymes,
oligosaccharides
Molecular recognition of carbohydrates by proteins plays
an important role in many intracellular and intercellular
processes such as the trafficking of proteins, bacterial and
viral infection, normal cell differentiation, tumor progres-
sion and metastasis.1 The biosynthesis of mammalian oli-
gosaccharides in vivo is mediated by glycosyltransferases
that sequentially transfer a single pyranosyl residue from
a sugar nucleotide donor to a growing carbohydrate chain
acceptor. Many successful syntheses using modified ac-
ceptor structures and the natural sugar nucleotides have
been reported. In comparison only a few reports deal with
the use of unnatural sugar nucleotides.2 In order to mea-
sure the activity of glycosyltransferases a variety of assays
have been developed over the years.3 The activity of gly-
cosyltransferases is usually determined using radiochem-
ical assays,3 ELISA assays,4 coupled enzyme assays5 and
recently capillary electrophoresis (CE) with laser induced
fluorescence detection.6 While these methods are well es-
tablished, there is a need for sensitive assays amenable to
high throughput format for screening. To search for sensi-
tive methods to determine glycosyltransferase activity we
became interested in developing a glycosyltransferase as-
say based on internal fluorescence quenching. Internal
fluorescence quenching7 assays have previously been
used to determine the enzymatic activity of hydrolytic en-
zymes such as peptidases and proteases8 and DNA-join-
ing reactions.17 The assay requires a substrate bearing a
fluorophoric emitter and a fluorophoric quencher on each
side of the cleavable bond. Enzymatic cleavage of the
molecule is followed by release of the fluorescence as the
proximity of the two chromophores decreases. It appeared
to us that the same principle could be applied on glycosyl-
transferase substrates provided that the emitter and
quencher pair is connected through a glycosyldiphosphate
The synthesis of the uridine-modified portion of the sugar
nucleotide containing the quencher is outlined in
Scheme 1. Palladium-catalyzed oxidative coupling of
2¢,3¢-O-isopropylideneuridine with methyl acrylate in ac-
etonitrile afforded 5-substituted uridine 5 in 70% yield.10
Hydrogenation of the double bond using Pearlman’s cata-
lyst and ester hydrolysis (0.5 M KOH) provided the acid
6. Compound 6 was then used to install the quencher moi-
ety. This was achieved through coupling of the amine 11
(prepared in a two step procedure from commercially
available Fmoc-3-nitro-Tyr-OH) with the acid 6 to yield
the amide 7. Phosphorylation of the 5¢-positon of ribose
was achieved through reaction with phosphorus oxychlo-
ride in triethylphosphate11 followed by deblocking of the
isopropylidene protecting group (2 N HCl/H2O) and ion
exchange to afford the phosphate 8. Compound 8 was ac-
tivated as the nucleoside 5¢monophosphomorpholidate 9
by treatment with 1,3-dicyclohexylcarbodiimide and mor-
pholine in tert-butyl alcohol at elevated temperature. Sub-
sequently, the activated nucleoside monophosphate was
coupled to the galactose 1-phosphate 19 (Sigma) under
1H-tetrazole-catalyzed conditions12 to furnish the uracil-
modified sugar nucleotide 2 in 33% yield.
SYNLETT 2004, No. 10, pp 1784–1788
0
9
.0
8
.2
0
0
4
Advanced online publication: 15.07.2004
DOI: 10.1055/s-2004-829563; Art ID: S03404ST
© Georg Thieme Verlag Stuttgart · New York

LETTER
Synthesis of Internally Fluorescence-Quenched Sugar Nucleotides
1785
NH2
O
O
O
H
N
H
COOEt
b
H
O
N
H
N
O
N
O
O
O
N
O
HO
HO
H
N
H
N
O
N
N
O
O
O
a
HO
HO
O
O
O
HO
O P O P O
O2N
O
O
OH
O
O
O
O
HO OH
Na
Na
1
5
4
H
N
O
O
O
HN
O
OH
O
HO
HO
H
N
O
O
N
H
O
N
H
N
H
O
O
N
O
N
H
N
COOH
HO
N
O P O P O
O
O2N
O
O
O
HO
O
OH
O
HO
O2N
O
HO OH
Na
Na
c
OH
2
O
O
O
O
NH2
O
N
H
6
O
N
7
O
HO
HO
H
N
O
O
d
O
O
O
HO
O P O P O
O
O
NH
OH
O
O
O
N
HO OH
Na
Na
H
N
H
3
N
O
Figure 1 Molecular structure of modified UDP-Gal analogues 1-3.
O
O
O
P
O
X
O2N
R1
The synthesis of the galactose-modified portion of the
sugar nucleotide containing the emitter moiety is outlined
in Scheme 2. O-cyanomethylation13 of 1,2;3,4-di-O-
HO
OH
isopropylidene-D-galactopyranose
(NaH,
MeCN,
X = Na
8: R1 = O , Na ;
BrCH2CN) afforded the cyano compound 13 in 81%
yield. Compound 13 was effectively reduced to the amine
using the borane-methyl sulfide complex in tetrahydrofu-
ran before being converted into the hydrochloride salt 14.
Compound 14 was then used to install the emitter moiety
by coupling to Fmoc-protected o-amino benzoic acid.
This was achieved by using 2-(1H-benzotriazole-1-yl-
1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) as
coupling reagent to afford the amide 15 in 83% yield. De-
blocking of the galactose portion (80% HOAc, 90 °C) fol-
lowed by acetylation and selective deacetylation (BnNH2,
THF) of the anomeric hydroxyl group provided the hemi-
acetal 16 in 52% overall yield after three steps. The phos-
phate group was installed by treatment of 16 with lithium
bis(trimethyl-silyl)amide (1 M solution in hexane) in THF
at –78 °C followed by trapping of the intermediate anion
with dibenzylphosphoryl chloride to produce the phos-
phate ester 17 in 50% yield. Deblocking of the compound
was achieved by a three-step procedure. First, the benzyl
protecting groups on the phosphate were removed by hy-
drogenation using Pearlman’s catalyst followed by treat-
ment with 20% piperidine in DMF to ensure complete
cleavage of the Fmoc-protecting group and purification
e
X = dicyclohexyl-
;
carboxamidinium
N
O
9: R1
=
OH
O
HO
HO
f
HO
OPO3H2
.
(C8H17)3N
2
19
H
OH
NO2
O
N
g
Fmoc-3-nitro-Tyr-OH
H2N
10
11
Scheme 1 Synthesis of UDP-Gal analogue 2. Reaction conditions:
(a) Pd(OAc)2, CH2CHCOOEt, MeCN (70%); (b) 1. H2/Pd(OH)2; 2.
KOH (92%); (c) 11, TBTU, DMF, DIEA (78%); (d) 1. POCl3,
PO(OEt)3; 2. HCl (90%); (e) DCC, morpholine, tert-butanol/H2O;
1:1, 95 °C (89%); (f) 19, 1H-tetrazole (33%); (g) 1. TBTU, DMF,
CH3(CH2)2NH2; 2. 20% piperidine/DMF (80%).
Synlett 2004, No. 10, 1784–1788 © Thieme Stuttgart · New York

1786
F. Schweizer et al.
LETTER
by chromatography. The remaining acetate protecting
groups were cleaved by exposure to a ternary solvent mix-
ture (Et3NH–MeOH–H2O 1:3:1) to produce the galactose-
modified emitter moiety, which was subsequently con-
verted into the sodium form 18 for stability reasons. The
pyridinium salt of monophosphate 18 was then coupled to
UMP (uridine 5¢-monophosphomorpholidate 4-morpholi-
no-N,N¢-dicyclohexylcarboxamidine salt, Sigma) in pyri-
dine to afford the emitter-modified UDP-Gal analogue 3
in 40% yield. We then applied the same coupling condi-
tions to couple the uridine-modified quencher moiety 9
with the pyridinium salt of 18 to produce the target com-
pound 1 in an overall yield of 21% yield.
NC
O
O
OH
O
O
O
O
i
O
h
O
O
O
O
13
12
FmocHN
O
O
NH
O
H3N
Cl
j
O
O
O
O
O
O
With UDP-Gal analogues 1–3 in hand we then studied
their use as potential substrates for galactosyl transferase
reactions. The enzymes selected were as follows: (1)
blood group B a(1→3) galactosyltransferase [blood group
B a(1→3) GalT E.C. 2.4.1.37],14 which catalyzes the
transfer of D-galactopyranose to from UDP-Gal to the 3-
hydroxyl group of a-L-Fucp-(1→2)-b-D-Galp-OR; (2)
a(1→3) galactosyltransferase,15 which catalyzes the
transfer of D-galactopyranose from UDP-Gal to the 3¢-hy-
droxyl group of b-D-Galp-(1→4)-b-D-Glcp-OR; (3) milk
bovine b(1→4) GalT,16 which catalyzes the transfer of D-
galactopyranose from UDP-Gal to the 4-hydroxyl group
of Glcp(NAc)-OR. Initially, we studied the transfer reac-
tions of the UDP-Gal analogues 1–3 with the substrates
20–22 shown in Scheme 3. In all cases, except for the
milk b(1→4) GalT-catalyzed reactions of donors 3 and 1
with acceptor 22, we observed transfer of the galactose
unit from the UDP-Gal donor analogues 1–3 to the accep-
tors 20–22 as evidenced by MALDI-TOF analysis of the
reaction products. In the case of UDP-Gal analogue 2, we
were able to monitor and measure (see Table 1) the rela-
tive rate of transfer when compared to unmodified UDP-
Gal by TLC. For all other transfer reactions TLC quanti-
fication was not possible due to the small amount of prod-
uct formed during the transfer reaction. Due to the low
transfer rates of UDP-Gal modified donors 1–3, no other
quantification methods such as CE and fluorescence-
quenching were attempted.
O
O
O
O
15
14
k
FmocHN
O
FmocHN
O
N
H
N
H
O
AcO
l
O
AcO
AcO
O
AcO
O
AcO
O
P
OH
AcO
BnO
O
16
17
BnO
m
O
NH2
O
HO
HO
n
N
H
1
O
HO
OPO3Na2
18
FmocHN
O
In conclusion, we have developed a synthetic route for the
synthesis of galactose-, uracil-modified UDP-Gal ana-
logues as well as an internally quenched UDP-Gal ana-
logue. Most of the UDP-Gal analogues are recognized by
three selected galactosyltransferases and permit transfer
of their galactose unit to the acceptor.
OH
o
26
3
Scheme 2 Synthesis of UDP-Gal analogues 1 and 3. Reaction con-
ditions: (h) MeCN, NaH, CH2BrCN (81%); (i) 1. BH3·Me2S, THF; 2.
HCl (75%); (j) 26, TBTU, DIEA, DMF (83%); (k) 1. 80% HOAc, 80
°C; 2. Ac2O, pyridine; 3. BnNH2, THF (52%); (l) LiHMDS, THF,
POCl(OBn)2, –49 °C (50%); (m) 1. Pd(OH)2, H2; 2. 20% piperdine/
DMF; 3. Et3NH–MeOH–H2O 1:3:3; 4. ion exchange (78%); (n) 9,
1H-tetrazole, pyridine (21%); (o) uridine 5¢-monophosphomorpholi-
date-4-morpholino-N,N¢cyclohexylcarbox-aminidine salt, pyridine,
1H-tetrazole (40%).
Table 1 Relative Rates of Transfer of Donor 2 with Acceptors 20–
22 Compared to Unmodified UDP-Gal
Enzyme
Donor
vrel (%)
Calf thymus a(1→3) GalT
Blood group B a(1→3) GalT
Milk bovine b(1→4) GalT
2
2
2
1
1
3
Synlett 2004, No. 10, 1784–1788 © Thieme Stuttgart · New York

LETTER
Synthesis of Internally Fluorescence-Quenched Sugar Nucleotides
1787
R1
O
General Procedure for the Synthesis of UDP-Gal Analogues 1–
3.
R1
O
OH
O
OH
O
HO
HO
OH
O
HO
HO
HO
O
Disodium-a-D-galactosyl phosphate analogue 18 (65 mg, 139
mmol) was passed through a BIO Rad AG 50W-X2 cation exchange
column (pyridinium form, 1.5 × 5 cm) and the solution was concen-
trated and co-distilled (2 × 8 mL) with dry pyridine. Uridine 5¢-
monophosphomorpholidate-4-morpholino-N,N¢-dicyclohexylcar-
boxamidinium salt analogue 9 (191 mg, 189 mmol) was added and
the mixture was co-evaporated with dry pyridine (3 × 6 mL). 1H-
Tetrazole (35 mg, 447 mmol) and dry pyridine (2 mL) were added,
and the solution was stirred at r.t. The reaction was monitored by
TLC [(2:1 i-PrOH–NH4OAc (1 M)] and NMR by removing small
aliquots (50 mL) from the reaction mixture after 12 h, 48 h, 72 h and
96 h. After 20 h (compound 3) and 120 h (compound 1) the mixture
was diluted with H2O (3 mL) and evaporated. The residue was sus-
pended in 4 mL of 100 mM NH4HCO3 and extracted with Et2O (3
mL). After evaporation, the residue was purified on a Bio-Gel P-2
column (2 × 65 cm), eluted with 100 mM NH4HCO3. The product-
containing fractions were concentrated, re-dissolved in H2O and
further purified using gradient reverse phase column chromato-
graphy on C-18 silica (H2O–H2O (50%)/MeOH). The product-con-
taining fractions were concentrated and the material was further
purified by repeating the previously described Bio-Gel P-2 column
separation followed by a final reverse phase column chromato-
graphy on C-18 silica as described earlier. Lyophilization of the
sample provided the desired target molecules 1 and 3.
O
OH
OH
21
O
OH
OH
OH
OH
HO
HO
O
O
R1
NHAc
22
20
O
R2
HO
O
R1
O
HO
OH
HO
O
HO
O
p
O
20
O
OH
OH
OH
23
HO
O
O
R2
OH
Procedure for the Synthesis of UDP-Gal Analogue 2.
Dipotassium-a-D-galactosyl phosphate analogue 18 (42 mg, 125
mmol) was passed through a BIO Rad AG 50W-X2 cation exchange
column (pyridinium form, 1.5 × 5 cm) and the solution was concen-
trated and co-distilled (2 × 8 mL) with dry pyridine. Trioctylamine
(55 mL, 125 mmol) and dry pyridine (5mL) was added, concentrated
and the mixture was co-evaporated with dry pyridine (3 × 6 mL) to
form 19. Uridine 5¢-monophosphomorpholidate-4-morpholino-
N,N¢-dicyclohexylcarboxamidinium salt analogue 9 (191 mg, 189
mmol) was added and the mixture was co-evaporated with dry pyri-
dine (3 × 6 mL). 1H-Tetrazole (30 mg, 426 mmol) and dry pyridine
(2 mL) were added and the solution was stirred at r.t. The reaction
was monitored by TLC [2:1 i-PrOH–NH4OAc (1 M)]. After 20 h,
the mixture was diluted with H2O (2 mL) and evaporated. The resi-
due was suspended in 4 mL of 100 mM NH4HCO3 and extracted
with Et2O (3 mL). After evaporation, the residue was purified on a
Bio-Gel P-2 column (2 × 65 cm), eluted with 100 mM NH4HCO3.
The product-containing fractions were concentrated, dissolved in
H2O and further purified using gradient reverse phase column chro-
matography on C-18 silica [H2O–H2O (50%)/MeOH] before ion
exchanged with a BIO Rad AG 50W-X2 cation exchange column
(sodium form, 1.5 × 5 cm) and lyophilized to provide the UDP-Gal
analogue 2 (39.4 mg, 33% yield).
OH
O
O
O
HO
HO
O
O
HO
O
q
R1
OH
21
OH
24
O
R2
OH
O
HO
HO
r
O
O
O
R1
22
HO
OH
NHAc
25
H2N
O
R1 = (CH2)CONH(CH2)2NH
R2 = (CH2)2NH
O
CO2
General Procedure for the Galactosyltransferase-Catalyzed
Reactions.
a(1→3) GalT Assay:
Me
N
Me
O
N
a(1→3) GalT was isolated according to the literature procedure.15
UDP-Gal analogues (1–3, 20 nmol) and lactose-TMR (21, 20 nmol)
were incubated with calf thymus a(1→3) GalT (80 mU) in buffer A
(10 mL) at r.t. (buffer A: 35 mM sodium cacodylate, 20 mM MnCl2,
0.1% Triton X-100, 0.8 mg/mL BSA, pH 6.5). The progress of the
reactions was monitored by TLC (65:35:7 CHCl3–MeOH–H2O)
and compared to the reaction of unmodified UDP-Gal (Sigma, 20
nmol) and lactose-TMR (21, 20 nmol) incubated with calf thymus
a(1→3) GalT (800 mU). Aliquots (1 mL) were taken after 2, 5, 8,15,
29, 25, 55, 100, and 150 min. Starting materials and products were
separated by TLC (65:35:7 CHCl3–MeOH–H2O). Relative transfer
rates were measured by integration of the new product spots using
SNAPSCAN and compared with unmodified UDP-Gal. Observed
Me
Me
Scheme 3 Glycosyltransferase-catalyzed reactions of UDP-Gal
analogue 1 with acceptors 20–22 to products 23 and 24. (p) 1, blood
group B a(1→3)GalT; (q) 1, a(1→3)GalT; (r) 1, milk b(1→4)GalT.
Synlett 2004, No. 10, 1784–1788 © Thieme Stuttgart · New York

1788
F. Schweizer et al.
LETTER
MS data (MALDI-TOF) for substrate 21 reacting with donor 1–3:
(D2O, H3PO4): d = –12.6 (d, JP,P = 20.8 Hz, P attached to galactose),
–11.2 (d, JP,P = 20.8 Hz, P attached to uridine). HRMS (ES) [M +
3Na]+ = 954.1411.
[M + H]+ = 1277.6; [M + H]+ = 1115.5; [M + H]+ = 1277.6.
Blood Group B a(1→3) GalT Assay:
1
Compound 3: H NMR (D2O): d = 3.52–3.62 (m, 2 H), 3.70–3.82
Recombinant blood group B a(1→3) GalT were prepared as previ-
ously described.14a–c UDP-Gal analogues (1–3, 20 nmol) and Fuc-
Gal-TMR (20, 20 nmol) were incubated with blood Group B
a(1→3) GalT (22 mU) in buffer B (10 mL) at r.t. (buffer B: 50 mM
sodium cacodylate, 20 mM MnCl2, 1 mg/mL BSA, pH 6.8). The
progress of the reactions was monitored by TLC (65:35:5 CHCl3–
MeOH–H2O) and compared to the reaction of unmodified UDP-Gal
(Sigma, 20 nmol) and Fuc-Gal-TMR (20, 20 nmol) incubated with
blood group B a(1→3) GalT (220 mU). Aliquots (1 mL) were taken
after 2, 5, 8,15, 29, 25, 55, 100, and 150 min and starting materials
and products were separated by TLC (65:35:5 CHCl3–MeOH–
H2O). Relative transfer rates were measured by integration of the
new product spots using SNAPSCAN and compared with unmodi-
fied UDP-Gal. Observed MS data (MALDI-TOF) for substrate 20
reacting with donor 1–3: [M + H]+ = 1261.6; [M + H]+ = 1099.5; [M
+ H]+ = 1261.6
(m, 5 H), 3.90 (dd, 1 H, J = 10.2 Hz, J = 3.2 Hz, H-3 galactose),
4.01 (d, 1 H, J = 3.2 Hz, H-4 galactose), 4.15–4.27 (m, 3 H), 4.29
(t, 1 H, J = 6.3 Hz, H-5 galactose), 4.31–4.35 (m, 2 H), 5.62 (dd, 1
H, J = 3.5 Hz, JH1,P = 7.3 Hz, H-1 galactose), 5.92 (d, 1 H, J = 5.6
Hz, H-1 ribose), 5.93 (d, 1 H, J = 8.1 Hz, uridine), 6.82–6.90 (m, 2
H, 2 × Ar-H), 7.32 (t, 1 H, J = 7.7 Hz, Ar-H), 7.45 (d, 1 H, Ar-H),
7.91 (d, 1 H, J = 8.1 Hz, uridine). 31P NMR (D2O, H3PO4): d =
–12.6 (d, JP,P = 18.9 Hz, P attached to galactose), –11.0 (d,
JP,P = 18.9 Hz, P attached to uridine). HRMS (ES): [M + Na]+ =
751.1239.
Acknowledgment
Financial support of this project was provided by NSERC.
References
Milk Bovine b(1→4) GalT Assay:
(1) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 322.
(2) Elhalabi, J. M.; Rice, K. G. Curr. Med. Chem. 1999, 6, 93.
(3) (a) Sadler, J. E.; Beyer, T. A.; Oppenheimer, C. L.; Paulson,
J. C.; Prieels, J.-P.; Rearick, J. I.; Hill, R. L. Methods
Enzymol. 1982, 83, 458. (b) Palcic, M. M.; Sujino, K.
Trends in Glycoscience and Glycotechnology 2001, 13, 361.
(4) Keshvara, L. M.; Gosselin, S.; Palcic, M. M. Glycobiology
1993, 3, 416.
(5) Gosselin, S.; Alhussaini, M.; Streiff, M. B.; Takabayashi,
K.; Palcic, M. M. Anal. Biochem. 1994, 220, 92.
(6) See: Sujino, K.; Uchiyama, T.; Hindsgaul, O.; Seto, N. O. L.;
Wakarchuk, W. W.; Palcic, M. M. J. Am. Chem. Soc. 2000,
122, 1261; and references therein.
(7) Yaron, A.; Carmel, A.; Katchalski-Katzir, E. Anal. Biochem.
1979, 95, 228.
(8) (a) Mayatoshi, E. D.; Wang, G. T.; Krafft, G. A.; Erickson,
J. Science 1990, 247, 954. (b) Meldal, M.; Breddam, K.
Anal. Biochem. 1991, 195, 141.
(9) Bergstrom, D. E. Nucleosides, Nucleotides Nucleic Acids
1982, 1, 1.
(10) Hirota, K.; Isobe, Y.; Kitade, Y.; Maki, Y. Synthesis 1987,
Milk Bovine b(1→4) GalT was prepared according to the previous-
ly published procedure.16 UDP-Gal analogues (1–3, 20 nmol) and
GlcNAc-TMR (22, 20 nmol) were incubated with blood Group B
a(1→3) GalT (44 mU) in buffer B (10 mL) at r.t. (buffer B: 50 mM
sodium cacodylate, 20 mM MnCl2, 1 mg/mL BSA, pH 6.8). The
progress of the reactions was monitored by TLC (65:35:7 CHCl3–
MeOH–H2O) and compared to the reaction of unmodified UDP-Gal
(Sigma, 20 nmol) and GlcNAc-TMR (22, 20 nmol) incubated with
milk bovine b(1→4) GalT (440 mU). Aliquots (1 mL) were taken af-
ter 2, 5, 8,15, 29, 25, 55, 100, and 150 min and starting materials and
products were separated by TLC (65:35:7 CHCl3–MeOH–H2O).
Relative transfer rates were measured by integration of the new
product spots using SNAPSCAN and compared with unmodified
UDP-Gal. Observed MS Data (MALDI-TOF) for substrates 22 and
donor 2 reacting with milk bovine b(1→4) GalT: [M + H]+ = 994.5.
Analytical Data for Compounds 1, 2 and 3.
1
Compound 1: H NMR (D2O): d = 0.66 (t, 3 H, -CH2CH2CH3),
1.30–1.40 (m, 2 H, -CH2CH2CH3), 2.50–2.68 (m, 4 H, -CH2CH2CO2NH-),
2.93–3.14 (m, 4 H, 2 × ArCH2-, 2 × CONHCH2-), 3.50–3.60 (m, 2
H), 3.72–3.77 (m, 4 H), 3.78–3.82 (m, 1 H, H-2 galactose), 3.91 (dd,
1 H, J = 10.2 Hz, J = 3.2 Hz, H-3 galactose), 4.00 (dd, 1 H, J = 3.2
Hz, J < 1 Hz, H-4 galactose), 4.18–4.26 (m, 3 H), 4.28–4.33 (m, 2
H, H-5), 4.33–4.36 (t, 1 H, J = 5.2 Hz, H-4 ribose), 4.40–4.44 {m,
1 H, Ha[Tyr(NO2)]}, 5.66 (dd, 1 H, JH1,H2 = 3.7 Hz, JH1,P = 7.3 Hz,
H-1 galactose), 5.93 (d, 1 H, J = 5.2 Hz, H-1 ribose), 6.80 (m, 1 H,
J = 7.9 Hz, J < 1 Hz, Ar-H), 6.85 (d, 1 H, J = 7.9 Hz, Ar-H), 7.10
(d, 1 H, J = 8.6 Hz, Ar-H), 7.29 (m, 1 H, J =1.4 Hz, J = 8.3 Hz, Ar-
H), 7.43 (m, 1 H, J = 1.2 Hz, J = 7.8 Hz, Ar-H), 7.51 (m, 1 H,
J = 2.1 Hz, J = 8.6 Hz, Ar-H), 7.62, (s, 1 H, Ar-H), 7.92 (m, 1 H,
J = 2.1 Hz). 31P NMR (D2O, H3PO4): d = –12.4 (d, JP,P = 20.5 Hz,
P attached to galactose), –11.1 (d, JP,P = 20.5 Hz, P attached to
uridine). HRMS (ES): [M + 2Na]+ = 1094.2386.
495.
(11) Yoshikawa, M.; Kato, T.; Takenishi, T. Tetrahedron Lett.
1967, 5065.
(12) Wittmann, V.; Wong, C.-H. J. Org. Chem. 1997, 62, 2144.
(13) Malet, C.; Hindsgaul, O. J. Org. Chem. 1996, 61, 4649.
(14) (a) Seto, N. O. L.; Palcic, M. M.; Hindsgaul, O.; Bundle, D.
R.; Narang, S. A. Eur. J. Biochem. 1995, 234, 323.
(b) Seto, N. O. L.; Palcic, M. M.; Compston, C. A.; Li, H.;
Bundle, D. R.; Narang, S. A. J. Biol. Chem. 1997, 272,
14133. (c) Seto, N. O. L.; Compston, C. A.; Evans, S.;
Bundle, D. R.; Narang, S. A.; Palcic, M. M. Eur. J. Biochem.
1999, 259, 770. (d) Polakowski, M. S. L.; Seto, N. O. L.;
Leinala, E.; Borisova, S.; Blancher, A.; Roubinet, F.; Evans,
S. V.; Palcic, M. M. J. Biol. Chem. 2003, 278, 12403.
(15) Fang, Y. R.; Sujino, K.; Lu, A.; Gregson, J.; Yeske, R.;
Kamath, V. P.; Ratcliffe, R. M.; Shur, M. J.; Wakarchuk, W.
W.; Palcic, M. M. Carbohydrate Bioengineering,
Interdisciplinary Approaches, In Chemi-enzymatic
Synthesis of Toxin Binding Ligands; Teeri, T. T.; Svensson,
B.; Gilbert, H. J.; Feizi, T., Eds.; Royal Society of
Chemistry: Cambridge UK, 2002, 127–134.
1
Compound 2: H NMR (D2O): d = 0.66 (t, 3 H, -CH2CH2CH3),
1.30–1.40 (m, 2 H, -CH2CH2CH3), 2.50–2.68 (m, 4 H, -CH2CH2CO2NH-),
2.94–3.04 (m, 3 H, 2 × ArCH2-, CONHCH2-), 3.10–3.16 (m, 1 H,
-CONHCH2-), 3.72 (dd, 1 H, J = 4.9 Hz, J = 12.3 Hz, H-6a galac-
tose), 3.76 (dd, 1 H, J = 6.6 Hz, J = 12.3 Hz, H-6b galactose), 3.79–
3.83 (m, 1 H, H-2 galactose), 3.91 (dd, 1 H, J = 9.9 Hz, J = 2.4 Hz,
H-3 galactose), 4.00 (dd, J = 2.4 Hz, J ca. 1 Hz, H-4 galactose),
4.14–4.18 (m, 1 H, H-5 galactose), 4.20–4.30 (m, 3 H, 2 × H-5 ri-
bose, H-3 ribose), 4.34–4.39 (m, 2 H, H-2 ribose, H-4 ribose), 4.40–
4.44 {m, 1 H, Ha[Tyr(NO2)]}, 5.66 (dd, 1 H, JH1,H2 = 3.8 Hz,
(16) Barker, R.; Olsen, W. K.; Shaper, J. H.; Hill, R. L. J. Biol.
Chem. 1972, 247, 7135.
(17) Sando, S.; Kool, E. T. J. Am. Chem. Soc. 2002, 124, 2096.
(18) Washiya, K.; Furuike, T.; Nakajima, F.; Lee, Y. C.;
Nishimura, S.-I. Anal. Biochem. 2000, 283, 39.
JH1,P = 6.1 Hz, H-1 galactose), 5.93 (d, 1 H, J = 5.3 Hz, H-1 ribose),
7.00 (d, 1 H, J = 7.7 Hz, Ar-H), 7.43 (dd, 1 H, J = 7.7 Hz, J ca. 1
Hz, Ar-H), 7.66 (s, 1 H, Ar-H), 7.88 (d, 1 H, J = ca. 1 Hz). 31P NMR
Synlett 2004, No. 10, 1784–1788 © Thieme Stuttgart · New York